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Comparative Study
. 1999 Jan 5;96(1):79-84.
doi: 10.1073/pnas.96.1.79.

Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals

J Buck  1 , M L Sinclair, L Schapal, M J Cann, L R Levin
Affiliations
Free PMC article
Comparative Study

Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals

J Buck et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Mammals have nine differentially regulated isoforms of G protein-responsive transmembrane-spanning adenylyl cyclases. We now describe the existence of a distinct class of mammalian adenylyl cyclase that is soluble and insensitive to G protein or Forskolin regulation. Northern analysis indicates the gene encoding soluble adenylyl cyclase (sAC) is preferentially expressed in testis. As purified from rat testis cytosol, the active form of sAC appears to be a fragment derived from the full-length protein, suggesting a proteolytic mechanism for sAC activation. The two presumptive catalytic domains of sAC are closely related to cyanobacterial adenylyl cyclases, providing an evolutionary link between bacterial and mammalian signaling molecules.

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Figures

Figure 1
Figure 1
Purification of sAC. (A) Silver stained gel and (B) activity profile of selected fractions from Analytical QA column chromatography. Ten-microliter aliquots of selected fractions were separated on a 12% SDS/PAGE gel and silver stained. The first lane is low molecular weight silver stain marker, and each subsequent lane corresponds to the fraction assayed for sAC activity beneath it. Bars represent the average sAC activity in duplicate assays for each fraction. Error bars indicate the standard deviation from the mean.
Figure 2
Figure 2
sAC amino acid sequence. Predicted amino acid sequence of rat sAC. Amino acids in bold indicate presumptive catalytic domains, C1 and C2. Double-underlined amino acids correspond to sequences of tryptic peptides derived from the purified 48-kDa protein. Dotted underlined amino acids conform to a consensus P loop sequence, and underlined sequences are predicted to form a leucine zipper. Valine 469 is underlined and is the last sAC amino acid in the catalytically active heterologously expressed truncation.
Figure 3
Figure 3
sAC has two presumptive catalytic domains most closely related to bacterial AC catalytic domains. (A) Diagram of presumptive catalytic domains of sAC aligned with catalytic portions of various bacterial ACs. The relative similarities are “expect values” taken directly from a blast search of the sAC protein vs. the nonredundant GenBank database. These values estimate the statistical significance of the match by specifying the number of matches expected to occur by chance. Relative locations of the catalytic domains within the bacterial ACs are represented as shaded boxes (–26) and are aligned under the sAC presumptive catalytic domain with greater similarity. (B) Phylogenetic relationship between catalytic domains from a variety of ACs aligned by using clustalw (dna*) represented as an unrooted dendogram constructed by using fitch (phylip 3.5) (27) with Anabaena spirulina cyaC used as the outgroup. Accession numbers for the aligned sequences are AC1 (bovine Type I: M25579), AC9 (mouse Type IX: Z50190), Dd ACA (Dictyostelium ACA: Q03100), Dd ACG (Dictyostelium ACG: Q03101), Anabaena sp. cyaA (2126532), Anabaena sp. cyaB1 (1754638), Anabaena sp. cyaB2 (1754640), Anabaena sp. cyaC (2575807), Anabaena cy. cya (2126532), Stigmatella cyaA (729248), Stigmatella cyaB (729250), Sc CYR1 (Saccharomyces cerevisiae CYR1: M12057), and Sp CYR1 (Schizosaccharomyces pombe CYR1: M24942).
Figure 4
Figure 4
sAC is a single copy gene preferentially expressed in testis. (A) Southern blots of 10 μg rat genomic DNA digested with BamHI (B), EcoRI (E), HindIII (H), and XhoI (X) probed at high (Left) or low (Right) stringency by using the 1-kb PCR-generated sAC fragment containing both presumptive catalytic domains. (B) Rat multiple tissue Northern blot (CLONTECH) representing approximately 2 μg poly-A+ RNA from testis (T), kidney (K), skeletal muscle (Sk), liver (Li), lung (Lu), spleen (S), brain (B), and heart (H) probed with 1-kb PCR-generated sAC fragment (Upper) or with actin control (Lower). The sAC transcript is approximately 5.3 kb and, in most tissues, actin is approximately 2.0 kb.
Figure 5
Figure 5
sAC is active in HEK293 cells. (A) Adenylyl cyclase activity in the presence of 5 mM MnCl2 or 5 mM MgCl2 is shown for cytosolic and particulate extracts from HEK293 cells transiently transfected with pBK-CMV vector (░⃞), full-length sAC gene (▨), or truncated sAC gene (▧). Values are expressed as pmols of cAMP formed per minute per mg protein and represent averages of triplicate determinations. Error bars indicate the standard deviation from the mean. Note the 20-fold difference in scale of the ordinate between left and right panels. (B) sAC activity in whole-cell sonicates from HEK293 cells transfected with full-length sAC (Left) or truncated sAC (Right) assayed in the absence of any activators (░⃞) or in the presence of 100 μM forskolin (▨) or 5 mM GTPγS (□). Values represent the averages of quadruplicate determinations with vector transfected HEK293 values for each condition subtracted. Error bars indicate the standard deviation from the mean.
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